A Comparative Evaluation of the Genotoxic Effects of Mobile Phone Radiation Using Buccal Micronucleus Assay

Statement of the Problem: Mobile usage has increased worldwide over the past two decades. There are conflicting reports about the carcinogenic effects of cell phone radiation on the oral mucosa. Micronucleus (MN) is considered a reliable marker for genotoxic damage. Purpose: This study aimed to identify the impact of mobile phone radiation on the MN frequency in oral mucosal cells. Materials and Method: In this descriptive-analytical study, 50 mobile phone users between the age group of 20–38 years were included. Samples were obtained from the right and left cheek mucosa of each subject (a total 100 cell samples). Every participant filled out a questionnaire about his or her cell phone usage habits. Additionally, personal information such as age, gender, and body mass index (BMI) were assessed. The Feulgen and Papanicolaou staining methods were used for staining of the cell samples. A total of 1000 cells in each sample were evaluated for MNs. Results: The mean number of MN in exposed and non-exposed mucosa by Feulgen method was 0.71±1.13 and 0.57±1.36, respectively. Also in Papanicolaou staining, the mean number of MN in the exposed mucosa and non-exposed mucosa was 6.94±6.61 and 6.54±6.88, respectively, but these differences were not significant (p> 0.05). The frequency of MN in non-specific DNA staining was significantly (5- to 6-fold) higher than DNA-specific staining. We observed no statically significant differences between MN frequency according to age, gender, BMI, and other cell phone usage habits (p> 0.05). Conclusion: This study showed that cell phone use does not cause genotoxic effects in the buccal mucosa in the oral cavity. Moreover, using non-specific DNA staining methods can increase the frequency of MN by more than 5- to 6-fold.


Introduction
Over the past two decades, the use of mobile phones has become almost universal [1]. The number of mobile phone users reached 4.93 billion in 2018 and increased to 5 billion in 2019 [2]. Radiofrequency radiation (RFR) is a type of electromagnetic radiation that varies from 3 kHz to 300 GHz. Most cell phones transmit RFR signals within the 800-900 and 1,800-2,200 MHz ranges [1].
The global growth of mobile phone usage has risen concerns about the dangers of electromagnetic waves associated with this technology [3]. There are two separate perspectives on the impact of radio waves on health, the first of which is due to the long conversations raising the heat of cell phones and the temperature of contact with the surrounding tissues. The second reason may be the non-thermal effects of waves from mobile phones and transmitter stations [4].
The biological consequences of exposure to radio fr-equencies are controversial [5]. Decades of research have yielded conflicting results; for instance, the results of some studies were reported the existence of a causal relationship between exposure to radiofrequency electromagnetic fields with an increased risk of glioma, meningioma, infertility, and deafness [6][7][8][9]. However, the findings of other studies could not confirm these results [10][11][12].
Micronuclei (MNs) are structures that contain chromosome fragments or whole chromosomes that are not incorporated into the nucleus of a daughter cell during cell division due to failure to bind to the spindle, and therefore, reflect aneugenic or clastogenic damage [13].
According to Bonassi et al. [14], they are considered as markers for the risk of cancer. The micronucleus (MN) test is one of the most common methods used to evaluate exposure to mutagens, carcinogens, and DNA damage [14][15][16][17]. It determines the presence of small nuclear bodies called micronuclei. The abundance of micronuclei in peripheral blood lymphocytes is associated with the risk of cancer and cardiovascular disease, making it a reliable marker [14,18].
MN can also be counted in exfoliated cells, especially in the oral mucosa [15]. The use of MN assay of buccal mucosa cells has various advantages, such as highly fast and convenient cell extraction. Moreover, it is especially suitable for determining the effects of RFR emitted by mobile phones because the oral cavity is in the exposure area and there is a possibility of genotoxic changes in this area [3,15]. Current reports about the effect of cell phones radiation on nuclear anomalies in oral mucosal cells are contradictory [3,[15][16]19]. Therefore, this study aimed to investigate the effects of exposure to cell phones radiation on the MN frequency in the epithelial cells of buccal mucosa in the oral cavity. The inclusion criteria were defined as individuals in the age limit of 20-40 years, without history of systemic disease, having radiation in the previous 2 months, and occupations in contact with chemicals. The exclusion criteria were defined as smokers, tobacco, and alcohol users and having any oral mucosal lesion.

Sample Collection and Staining
We collected two cell samples from the right and left cheeks of each subject, a total of 100 cell samples from the buccal mucosa exposed and buccal mucosa none/ less exposed. The cases that used their cell phones with the same frequency on both sides were excluded from the research.
According to the method proposed by Thomas et al. [20], the patients were first asked to rinse their mouth with water to remove food particles, debris, and saliva.
Subsequently, exfoliated cells of buccal mucosa were collected using a cotton swab and applying circular motion 20 times. A separate swab was used for each cheek.  [21][22] were used to detect MN as (1) a clear, smooth, and round perimeter suggesting a membrane, Chicago, IL) using the paired t-test, independent samples test, one-way Anova and Pearson correlation coefficient. p Value less than 0.05 was considered statistically significant.

Results
In the present study, collecting buccal mucosa cells from the right and left cheeks of 50 mobile phone users (a total 100 samples) were examined to determine the MN frequency using two staining methods. The subjects were in the age range of 20-38 years with a mean age of 25.13.87 years and 52% of them were males. It was revealed that most of the subjects (80%) used their right cheek more frequently for a cell phone conversation, which was considered as exposed mucosa, while in 20% of cases, who used their left cheek more often during conversations, the buccal mucosa of the left side was considered as the exposed mucosa. The subjects who used both cheeks with the same frequency to talk on cell phones were excluded from the study. Other details about the subjects are given in Table 1.
The mean numbers of MN in exposed and nonexposed mucosa by Feulgen method were 0.71±1.13 and 0.57±1.36, respectively; the difference was not statistically significant ( Table 2) (Figure 1). Also in Papanicolaou staining, the mean number of MN in the ex-posed mucosa and non-exposed mucosa was 6.94±6.61 and 6.54±6.88, respectively, which was not significant (Table 3) (Figure 1).
It can be seen that the MN frequency were significantly (~ 6-fold) higher in non-specific DNA (Papanicolaou) than DNA-specific (Feulgen) staining. In both staining methods an increase in the mean of MN frequency was observed in men, individual which used mobile phone for more than 5 h per week and more than 10 years, but this difference was not statistically significant (Tables 4-5). Moreover, the mean of MN frequency   Table 2: Micronucleus count in exposed and nonexposed Buccal mucosa by Feulgen staining

Micronucleus (Mean±SD) p
Exposed mucosa 0.71±1.13 0.459 Non exposed mucosa 0.57±1.36 Table 3: Micronucleus count in exposed and non-exposed Buccal mucosa by Papanicolaou staining Groups Micronucleus Mean±SD p Exposed mucosa 6.94± 6.61 0.285 Non exposed mucosa 6.54±6.88  was lower in subjects using headphones than in those who did not use headphones, which was not significant.
In the quantitative analysis of some variables (age, has not yet been resolved [2,15].
The oral cavity is located in the area exposed to cell phone radiation, and the epithelial tissue is a target tissue for carcinogenic lesions. On the other hand, the MN test on buccal mucosa is very reliable and widely used since not only can it detect DNA damage but also it is able to evaluate chromosomal instability and cell death [15]. In addition, it is a reliable marker for an abnormal cell cycle following ectopic mitosis [2]. This test is a sensitive, non-invasive, low-cost, fast, and easy technique, in which oral mucosal cells similar to different types of cells especially lymphocytes, do not need to be cultured [19].
In different studies, various wooden spatulas or brushes were utilized to extract and isolate the cells [16,19,23]. Nonetheless, in this research, cotton swabs were used since this method is more reliable for cell recovery and more convenient for participants [15]. In the present study, the mean MN frequency in the exposed and nonexposed buccal mucosa to mobile phone radiation was examined using two staining methods. According to the results of the current study, the mean number of MN in the exposed mucosa of the subjects was not significantly different from that in the non-exposed mucosa, which was consistent with the studies conducted by Hintzsche et al. [15], Ros-Lior et al. [19] Souza et al. [13], and de phones; using headphones helps keeping the phone away from the body and subsequently eliminate the direct effect of RF on the body and reduce the local temperature around the ear area [2]. Finally, they noted that using cell phones, even within an acceptable distance from the body, could cause genotoxicity when used for a long time [2]. Additionally, when the cell phone is used predominantly on one side of the face, it may cause more genotoxicity due to the increased radiation and heat [2]. In our study, the mean MN was lower in cases that used headphones than in those who did not use them; however, this difference was not statistically significant.
In Banerjee et al. [23] study, individuals divided into two groups of the less mobile users (less than 5 years with 3 hours/week) and high mobile users (more than 5 years with 10 hours/week), and determined the number of MN using acridine orange staining. Based on the results of this research, a significant increase of MN mean was reported in the high mobile users group. In addition, the mean MN was significantly lower in cases utilizing headphones. It was also revealed that the subjects who complained more about heat around their ears had a higher mean MN, indicating that heat had a strong synergistic effect possibly by activating heat shock proteins on genotoxic damage [23].
It has been reported that heat shock protein 70 increases the radioadaptive response [24]. Furthermore, a local increase in temperature may damage the mitochondrial membrane and lead to the release of cytochrome C and activation of caspases-3 and -9 [25]. In a study performed by Daroit et al. [3], the effect of cell phones was investigated on the cytogenic abnormalities of oral mucosal cells in different areas (i.e., lower lip, tongue border and mouth floor). Accordingly, a slight increase was observed in the number of micronucleated cells in the lower lip and binucleated cells in the mouth floor among subjects who used mobile phones for more than 60 min per week. It was also mentioned that exposure to cell phone electromagnetic radiation might be associated with the development of nuclear anomalies among individuals using cell phones more than 60 min/ week for more than 8 years [3]. Yadav  In this study, we did not find any statistically significant changes in MN frequency regarding age, gender, and BMI. Similar to our study, Hintzsche and Stopper [15] reported that no significant difference was observed in the frequency of MN considering the factors of gender, BMI, and smoking.
In the current study, the mean of MN frequency was not significantly different in the subjects according to duration of mobile phone use per week, and years of using mobile phones. In a study carried out by de Oliveira et al. [5], in agreement with the results of the present study, the mean MN scores were not significantly different in the subjects in terms of duration of daily cell phone use, and years of cell phone use.
In the present study, subjects under 40 years were selected for research purposes since aging is considered a risk factor for the frequency of MN [19]. In this respect, the quantitative analysis of variables in our study showed a significant difference in the relationship between an increase in the age and the mean of MN in the subjects examined using the Papanicolaou staining method. We suggest future studies with larger sample size on this subject.

Conclusion
According to the results of the present study, although an increase in the number of MN was observed in the exposed mucosa to mobile radiation compared to nonexposed mucosa, this difference was not statistically significant. The use of non-specific DNA staining (Papanicolaou) methods can increase the frequency of micronuclei by more than six times, therefore employing DNA-specific methods (Feulgen) in future studies is recommended. In addition, no genotoxic effects as a result of exposure to mobile radiation were observed in the oral mucosa in relation to any parameter.